Innate immune response to infectious agents or local cell damage constitutes a major mammalian defense mechanism. Inflammatory cytokines and interferons are produced, mainly by myeloid cells, in response to bacterial or viral infection and they limit the microbial infection by either acting directly on the infected cells or activating immune cells of the adaptive immune system. There are multiple cytoplasmic pattern recognition receptors for sensing cytoplasmic DNA produced by either infecting microbes or damaged nuclei or mitochondria. All of these receptors use STING as the common signaling adaptor protein. This proposal is for investigating two novel features of STING signaling that were revealed by our preliminary experiments. Although TRIF is a known adaptor protein for TLR3 and TLR4 signaling, we discovered that it promotes STING signaling as well. Our aim is to determine how TRIF functions in the STING signaling pathway and how it affects resistance against HSV-1, a virus that triggers the STING signaling pathway. We have also observed that, like several TLRs, STING requires tyrosine phosphorylation to trigger its signaling activity. Moreover, the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is required for STING Tyr phosphorylation and its ability to induce cytokine genes. Our second aim is to investigate the mechanism of EGFR-mediated STING Tyr phosphorylation and its role in downstream signaling. Our proposed studies will illuminate new features of STING signaling, which is essential for eliciting innate immune response to cytoplasmic DNA.